Rett syndrome (RTT) is a debilitating neuropsychiatric disorder caused by mutations in the X-linked methyl CpG binding protein 2 (MECP2) gene, which encodes for a protein with the same name (MeCP2). Many of the RTT-causing mutations create a truncating null allele, suggesting that RTT is due to a loss of MeCP2 function. Interestingly, overexpression of MeCP2 due to a duplication spanning MECP2 also causes a progressive neurological syndrome that shares many features with RTT, including cognitive deficits, autistic features, motor abnormalities, seizures, and stereotyped behaviors. Studies in mice that either lack (RTT model) or express 2X MeCP2 (duplication model) reveal that the loss and gain of MeCP2 has opposing effects on excitatory synapses and gene expression, suggesting that the duplication causes the disorder by a "hyperfunction" mechanism. However, the challenge has been to understand exactly what function of MeCP2 is being exaggerated when it is overexpressed. Traditionally, MeCP2 was believed to be a transcriptional respressor, yet gene expression data from the animal models of RTT and duplication syndrome raise a question about this model in the nervous system, as loss of MeCP2 results in decreased expression of the majority of genes altered in the hypothalamus, whereas its gain leads to increased expression of the same genes. Newer hypotheses raised the possibility that MeCP2 normally dampens transcriptional noise such that in its absence, increased basal transcription throughout the genome might lead to decreased expression of neuronal genes. This model does not quite explain why doubling the protein will enhance expression of so many activity dependent neuronal genes. Another hypothesis proposes that overexpression of MeCP2 might titrate co-repressors, thus resulting in an increase of gene expression. In this proposal, I aim to test these hypotheses by generating and characterizing mice that overexpress MECP2 alleles that disrupt either of its two key functional domains. I propose that MeCP2 requires both its key domains to mediate the duplication phenotypes. To this end, I will study the in vivo consequences of two different RTT-causing mutations: R111G, which abolishes methyl-CpG binding, and R306C, which is in the transcriptional repression domain and whose precise effect on protein function is currently unknown. I will fully characterize these alleles in the context of both overexpression and in a MeCP2 null background to gain insight into the function of the two domains. I will then investigate the molecular and biochemical consequences of these two mutations by examining gene expression changes and the mechanisms leading to these changes, including testing specific histone marks and potential interactors. The neurobehavioral and molecular studies will help us to determine how the R306C mutation affects the protein's function, will provide insight into the mechanism by which overexpression causes disease, and will pinpoint whether the mechanism involves hyperfunction or titration of normal binding partners.